Electrophoresis is a technique used to separate charged species on the basis of size, electric charge, and other physical properties. In electrophoresis, the charged species migrate through a conductive electrophoretic medium, which may be (but is not required to be) a gel, under the influence of an electric field. Activated electrodes located at either end of the electrophoretic medium provide the driving force for the migration. The properties of the molecules, including their charge and mass, determine how rapidly the electric field causes them to migrate through the electrophoretic medium.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionizable groups. Because of these ionizable groups, at any given pH, many important biological molecules exist in solution as electrically charged species. The electrically charged species enable doctors and scientists to separate nucleic acids and proteins using electrophoresis.
Separation of molecules, biological or otherwise, using electrophoresis depends on various forces, including charge and mass. When a biological sample, such as a protein or DNA, is mixed in a buffer solution and applied to an electrophoretic medium, these two forces act together. Separation using electrophoresis is possible because the rate of molecular migration through the electric field depends on the strength of the field, the charge, size, and shape of the molecules, and the ionic strength and temperature of the buffer through which the molecules are moving. During electrophoresis, the applied electrical field causes the molecules to move through the pores of the electrophoretic medium based on the molecular charge. The electrical potential at one electrode repels the molecules while the potential at the other electrode simultaneously attracts the molecules. The frictional force of the electrophoretic medium also aids in separating the molecules by size. Typically, after the applied electrical field has been removed, the molecules may be stained. After staining, the separated macromolecules can be seen in a series of bands spread from one end of the electrophoretic medium to the other. If these bands are sufficiently distinct, the molecules in these zones can be examined and studied separately by fixing macromolecules and washing the electrophoretic medium to remove the buffer solution.
Separating lipoprotein particles in bodily fluids (e.g., serum or plasma) provides information on the levels of various lipoprotein particles. Various disease states are linked to levels of apolipoproteins and/or lipoprotein particles including but not limited to cardiovascular disease, Alzheimer's disease, hyperlipidemia, abetalipoproteinemia, hypothyroidism, liver disease, diabetes mellitus, and renal problems. Higher levels of apolipoprotein B and LDL particles have been associated with increased risk of cardiovascular disease. It has been disclosed that differences in the amount of cholesterol in a particle may also play a role in the risk of cardiovascular disease. Small dense LDL, having more cholesterol ester, appears to be correlated with a higher risk of cardiovascular disease. However, increased levels of HDL correlate with a decrease in risk for cardiovascular disease. Thus, accurate predictors of the risk of an individual of developing various diseases related to lipoprotein particles are needed for research, diagnostic, and therapeutic purposes.
Current electrophoretic technologies for detection of apolipoproteins and lipoprotein particles cannot identify or discriminate the presence of apolipoproteins on multiple particles simultaneously. There is thus a need for a more efficient method of detecting apolipoproteins and lipoprotein particles in an electrophoretic matrix.
Existing strategies to measure, detect, and quantify lipid protein particles use non-specific protein dye to detect the fixed proteins in a gel, limiting specificity in the presence of coincident apolipoproteins on the fixed lipid particle and the variety of diagnostic measurements possible.
There is a need for an “in-situ” detection system which can affect all analytic operations efficiently within the single zonal electrophoretic matrix. All other electrophoresis associated systems require operations including but not limited to extra-gel sample preparation, pre-electrophoresis protein/apolipoprotein reduction and/or denaturation, off-gel or secondary gel post electrophoresis operations. A system and method that combines or avoids such steps has significant economic benefits.
This invention is directed to overcoming these and other deficiencies in the art.